GCN4 is a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level by the eIF-2alpha kinase GCN2. Phosphorylation of eIF-2 by GCN2 under amino acid starvation conditions stimulates GCN4 translation by inhibiting guanine nucleotide exchange on eIF-2 by eIF-2B, reducing the concentration of the ternary complex eIF-2/GTP/Met-tRNAiMet. Genetic and biochemical analysis has shown that the GCN3, GCD7, and GCD2 subunits of eIF-2B comprise a regulatory subdomain that mediates inhibition of eIF2B by phosphoryated eIF2. These proteins contain two 70-residue homologous segments that are critcally required for their regulatory functions. GCD10 is a component of the eIF-3 complex and gcd10 mutations are suppressed by extra copies of genes encoding Met-tRNAiMet (IMT) or the yeast homologue of La autoantigen (LHP1). gcd10 mutants accumulate low levels of Met- tRNAiMet, but normal levels of all other tRNAs, and extra copies of IMT or LHP1 restore Met- tRNAiMet to nearly wild-type levels in gcd10 mutants. GCD10 has been localized to the nucleus by immunofluorescence, and we propose that GCD10 interacts with Met-tRNAiMet in the nucleus and delivers it to eIF3 in the cytoplasm where it can be utilized in ternary complex formation. GCN1 and GCN20 comprise a protein complex required to activate GCN2 kinase function in starved cells. An N-terminal segment of GCN20 binds to a domain in GCN1 related to translation elongation factor 3 (EF-3). GCN20 contains ATP-binding cassettes found in membrane transporters; however, immunofluorescence shows that GCN1 is distributed throughout the cytoplasm, and both GCN1 and GCN20 bind to polyribosomes in an ATP-stimulated fashion. We propose that GCN1/GCN20 function on the ribosome to facilitate delivery of uncharged tRNA to GCN2. Thr residues located between kinase subdomains VII and VIII in GCN2 are required for kinase activity in vivo and are sites of autophosphorylation in vitro. Conserved Thr residues in the human eIF2alpha kinase PKR are also critcally required for PKR function in vivo. Fragments of GCN2 containing the protein kinase or C-terminal ribosome-binding domains can form homo- or heterodimers in vivo, and we suggest that binding of the C-terminal domain to the kinase moiety regulates GCN2 function. Binding of uncharged tRNA to a histidyl-tRNA synthetase-related domain in GCN2 would alter the interaction between the C-terminal and kinase domains in a way that stimulates GCN2 activity. The transcriptional activation domain of GCN4 contains 7 clusters of bulky hydrophobic amino acids essential for transcription in vivo. These sequences are also required for in vitro binding of GCN4 to TBP-associated factors TAFII60 and TAFII90, suggesting that GCN4 activates transcription by recruiting the TBP-containing complex TFIID to the promoter. Transcriptional repression of the ADE5,7 gene by adenine involves down-regulating complex formation by the BAS1 and BAS2 activator proteins at the promoter. Interaction with BAS2 stimulates a latent activation domain in BAS1. A BAS1 mutation has been obtained that appears to permit BAS1/BAS2 complex formation in adenine-replete cells.